Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that "kiss-and-run" mechanisms may also play a role. Here, we have examined the dynamics of exocytosis in pancreatic MIN6 beta cells by imaging lumen- (neuropeptide Y/pH-insensitive yellow fluorescent protein; NPY.Venus) or vesicle membrane-targeted fluorescent probes (synaptobrevin-2/enhanced green fluorescent protein; synapto.pHluorin, or phosphatase on the granule of insulinoma-enhanced green fluorescent protein, phogrin.EGFP) by evanescent wave microscopy. Unexpectedly, NPY.Venus release events occurred much less frequently (13%-40% maximal rate) than those of synapto.pHluorin, even though the latter molecule, but not phogrin.EGFP, usually diffused away from the site of fusion. Thus, the majority of exocytosis occurs in these cells by kiss-and-run events that involve either the release of small molecules only, small molecules and selected membrane proteins, or all soluble cargoes ("pure," "mixed," and "full" kiss-and-run, respectively). Changes in the activity of synaptotagmin IV, achieved here by overexpression of the wild-type protein, may allow different stimuli to alter the ratio of these events, and thus the release of selected vesicle cargoes.
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"Green/Yellow FP's with pKa>6 such as EGFP (pKa = 6) and pHluorin (pKa = 7.6) detect the luminal pH change during exocytosis and are therefore powerful tools to study membrane fusion and fission [9,10,24,30,34,66]. In (neuro)endocrine cells, dense core granules are large and sparse enough to be imaged individually and allow pH-sensitive FP's to be used to detect the opening of the exocytotic fusion pore and to measure its life time [9,19,35,61]. Since EGFP retains some of its fluorescence in intact granules, it is a preferred granule marker as it allows observation and tracking of the intact organelles before exocytosis. "
[Show abstract][Hide abstract]ABSTRACT: Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo-and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo-or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.
"With a pK a (i.e., the pH value at which the fluorescence intensity is 50% of maximal) of 7.2, SEP is nearly nonfluorescent at pH 5.5 (the pH of intracellular secretory vesicles and recycling endosomes) but brightly green fluorescent at pH 7.4 (the extracellular pH). The SEP protein, fused to relevant membrane proteins, has been extensively used to detect the exocytosis of synaptic vesicles, secretory vesicles, and recycling endosomes (Sankaranarayanan et al., 2000; Gandhi and Stevens, 2003; Tsuboi and Rutter, 2003; Yudowski et al., 2006; Balaji and Ryan, 2007; Jullié et al., 2014). Moreover, it has been used to detect the formation of clathrin-coated vesicles (CCVs) using the pulsed pH (ppH) protocol (i.e., alternating the extracellular pH between 7.4 and 5.5), which reveals the location of receptors that have been newly internalized with high temporal accuracy (Merrifield et al., 2005). "
[Show abstract][Hide abstract]ABSTRACT: Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.
Full-text · Article · Nov 2014 · The Journal of Cell Biology
"This analysis indicates that the calculated probability (∼2%) is much lower than the observed frequency (>10%,Figure 3G), suggesting that consecutive events are not randomly distributed events. Because fluorescence at the insertion site between consecutive events did not reach background levels (Figures 3B,D,F), we hypothesized that the fusion pore could close after an initial release of cargo and reopen for a consecutive release via a mechanism reminiscent of synaptic kiss-and-run (Stevens and Williams, 2000; Tsuboi and Rutter, 2003; Jaiswal et al., 2009; Alabi and Tsien, 2012). To test this hypothesis, we used the proton pump inhibitor bafilomycin to prevent reacidification of the closing vesicle after the initial release. "
[Show abstract][Hide abstract]ABSTRACT: Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.
Full-text · Article · Nov 2014 · Frontiers in Cellular Neuroscience